Fibrin sponge

ABSTRACT

The invention relates to a fibrin sponge comprising a residual moisture content of at least 3%, preferably of 3 to 35%, in particular 10 to 20%, and preferably containing a blood clotting activator or proactivator, a method of preparing this fibrin sponge as well as a kit for wound gluing which comprises the fibrin sponge and a component containing a blood clotting factor. The sponge according to the present invention is suitable for hemostasis, tissue adhesion and aiding wound healing.

The present invention relates to a fibrin sponge having a definedresidual moisture, methods of its preparation, its use for hemostasis,tissue adhesion and supporting wound healing as well as a kit for itsapplication.

In the prior art, the most varying hemostatic wound cover materials,also based on fibrin, e.g. fibrin sponges, fibrin membranes or fibringels, have been described and partially also used.

So far the opinion has prevailed that for hemostasis, a combination offibrinogen and thrombin or corresponding prothrombin activators isnecessary. Thus, e.g., fleece-like flat materials based on collagencomprising fibrinogen and thrombin (TachoComb®), flat materialscomprising fibrinogen and activated factor X (AT 0 359 653, IMMUNO AG)or flat materials comprising fibrinogen and thrombin as a drypreparation (EP 0 748 633, IMMUNO AG) have been known from the priorart. Whereas the fleece-like flat materials based on fibrinogen andactivated factor X are already soft and pliable, other flat materialsrequire absolutely dry storage so as to preclude a premature reaction ofthe fibrinogen contained therein with the thrombin or with other bloodclotting activators.

Bering E. A. jr. (J. Clin. Invest. Vol. 23, 1944, p. 586) discussesgeneral developments in the field of fibrin foams and their use ashemostatic agents.

A survey of various materials based on fibrin, e.g. fibrin powders,fibrin films and fibrin foams, is e.g. given in Gerendas M. “FibrinProducts as aids in hemostasis and wound healing”, pp. 277, in K. Laki(ed.) Fibrinogen, M. Dekker, New York, 1968.

Furthermore, fibrin foils or fibrin membranes, respectively, have beenknown from the prior art (cf. e.g. EP 0 485 210-A2). Such materials areextremely thin, non-absorbent and thus usable for hemostasis to alimited extent only. In contrast to fibrin sponges, these materials arecompressed on purpose during their preparation, e.g. by the applicationof weights. Therefore, these materials are primarily characterized bytheir high density, as compared to foams.

The present invention has as its object to provide a means forhemostasis, tissue adhesion and the aiding of wound healing based onfibrin which, compared to conventional means, has a good, in particularan improved, efficacy, is simple to handle with a simultanous simplecomposition that allows for an inexpensive preparation, and which alsohas a broad field of application. Therebeyond, it should be possible toreadily supply the fibrin material with other active and auxiliarysubstances.

According to the invention, this object is achieved in that a fibrinsponge having a residual moisture of at least 3%, preferably in therange of from 3 to 35%, in particular 10 to 20%, is provided which isstorage stable. The water content or the residual moisture,respectively, preferably is adjusted such that the fibrin sponge has asoft and smooth consistency, thereby substantially improving itshandling and efficacy.

From the prior art it has been known that dry preparations containingfibrin, which have a hard, brittle consistency, will become soft andsmooth (pliable) by increasing their residual moisture. However, therehas been a general prejudice as regards the stability of such proteinpreparations having an increased residual moisture, particularly if theycontain a sensitive enzyme, such as thrombin, or another activator orproactivator of blood clotting.

It is not without reason that such sensitive protein preparationscommonly are made stable by lyophilisation, a residual moisture of lessthan 3%, in most instances even of less than 1%, being attained.

Therefore, it has been suprising that a fibrin sponge preferably havinga content of thrombin and an increased residual moisture or an increasedwater content nevertheless is storage stable.

By storage-stable according to the present invention, a fibrin sponge isunderstood which even after a storage of 6 months, generally, however,even after a storage of 2 or more years at room temperature is stillextremely suitable for application, and the optionally present thrombinactivity or blood clotting activator or proactivator activity remainssubstantially preserved. Preferably, more than 70% of the activityremain over a period of at least 6 months, preferably 2 or more years,at room temperature. Even at a temperature of 370° C. the fibrin spongeaccording to the invention having a residual moisture of 15% has beenshown to be stable for at least 3 months, preferably more than 6 months,its thrombin activity being fully retained.

The preparation according to the invention particularly meets themanifold requirements made on a means for hemostasis, tissue adhesionand aiding wound healing, with the preparation according to theinvention, properties such as

a good and lasting efficacy, i.e. rapid hemostasis or adhesive effect,no secondary bleedings or late detachment of glued tissue parts,

a good tolerance even with multiple applications (no sensitisation)

complete resorption during the wound healing process,

smooth and undisturbed wound healing,

the greatest possible safety as regards the transmission of viral orother pathogens (such as, e.g., prions, the pathogens of bovinespongiform encephalitis, BSE),

simple use as well as the already mentioned

storage stability of the ready-to-use product can, e.g., be ensured.

Preferably, the fibrin sponge according to the invention is a fibrinfleece-like flat material or a fibrin foam having a low density and ahigh absorptive capacity.

According to the invention, by fibrin fleece-like flat material a fibrinsponge is to be understood which has a porous, in particular fine porousstructure substantially not interspersed by air bubbles, as it might beobtained by lyophilising non-foamed fibrin clots according to theinvention. By fibrin foam, on the other hand, a material is to beunderstood which is obtained by foaming a fibrinogen solution, admixingwith a thrombin solution and lyophilising the thus obtained foamedfibrin clot. Foaming may also occur immediately after the addition ofthe thrombin.

The fibrin sponge according to the invention may also be used as such.It may essentially consist of fibrin and the thrombin required forproducing the fibrin from fibrinogen, and surprisingly even this simpleembodiment exhibits an excellent hemostatic and adhesive effect.

It may, however, be impregnated with further additives, preferably ablood clotting activator or proactivator. Such additives are preferablyhomogenously distributed in the preparation according to the invention.

The blood clotting activator or proactivator preferably is selected fromthe group consisting of thrombin, prothrombin, activated factor X,prothrombin complex, activated prothrombin complex, FEIBA, calcium ionsand mixtures thereof.

In particular, additional thrombin may be incorporated into thepreparation according to the present invention, preferably in an amountof between 1 and 300 U/cm³, more preferably between 5 and 100 U/cm³,most preferably between 10 and 50 U/cm³.

Surprisingly, it has been found that the preparations according to theinvention based on solid fibrin also with an additional content ofthrombin are still stable over long periods of storage at refrigeratoror room temperature and even at 37° C. (in an accelerated stabilitytest). In particular, the thrombin activity contained is substantiallymaintained over long periods of time. The contained thrombin activitymay, e.g. after extraction with 1 M NaCl solution, be determinedaccording to methods known per se, e.g. with a chromogenic substrate(Th-1, Pentapharm).

In addition to substances, such as blood clotting activators orproactivators, the fibrin sponge according to the invention may alsocontain stabilizers, preservatives, antibiotics, therapeutic agents,antifibrinolytic agents, growth factors, further plasma proteins,enzymes, inhibitors or mixtures of such agents.

As stabilizers, preferably thrombin stabilizers, in particular polyoles,polysaccharides, polyalkylene glycols, amino acids or mixtures thereof,or substances stabilizing the water content, in particularpolysaccharides or polyoles, are used. The use of sorbitol, glycerol,polyethylen glycol, polypropylene glycol, mono- or disaccharides, suchas glucose or sucrose, or any sugar or amino acid which stabilizes thethrombin activity or the residual moisture, respectively, is preferredthere.

Likewise, the desired softness of the preparation may be attained orimproved by incorporating substances, e.g. hydro-stabilizing activesubstances, such as glycerol. After lyophilizing in the presence of suchsubstances, increased residual moisture levels can be obtained.

A preferred antifibrinolytic agent is selected from the group consistingof aprotinin, aprotinin derivative, α-2-macroglobulin, α-2-plasmininhibitor, α-1-plasmin inhibitor, plasminogen activator inhibitor,inhibitor or inactivator of activated protein C, a plasmin-bindingsubstance, or mixtures of these substances. Also synthetic substances,in particular synthetic fibrinolysis inhibitors, e.g. ε-amino-caproicacid or tranexamic acid, may be used. Also non-plasmaticantifibrinolytics, of vegetable origin, e.g., may be used according tothe invention.

Preferred growth factors are, e.g., cytokines. As further plasmaproteins preferably albumin, factor XIII or fibronectin are provided.

Furthermore, also specific proenzymes, enzymes or enzyme inhibitors maybe admixed with the fibrin sponge according to the invention, e.g. so asto regulate the absorption of the fibrin sponge in the body, i.e. eitherto accelerate or to inhibit it. Among these substances are, e.g.,plasminogens, collagenase, plasminogen activators, plasminogen activatorinhibitors, plasmin inhibitors or elastase inhibitors.

The preparations according to the invention as such already have a goodabsorptive capacity. Preferably, a slight amount of a tenside, such asTween® 80 (polyoxyethylene sorbitan monooleate) is additionallyincorporated, whereby the absorptive capacity of the preparationaccording to the invention may additionally be increased.

The preparation according to the invention thus is particularlycharacterized by the following properties:

It has a great softness or adaptability so that it can be applied to anunevenly formed wound surface without any problems. Furthermore, it hasa high absorptive capacity, whereby the emerging blood can be taken upwithout any delay.

Preferably, the fibrin sponge according to the invention has a liquidabsorption capacity of at least the 2-fold, preferably of more than the4-fold, of its own weight.

In a preferred embodiment, the material has a homogenous distribution ofits coagulation-promoting properties of such a level that the sucked-upblood clots in the preparation according to the invention within a fewseconds, preferably within less than 30 seconds.

The present invention also relates to a method of preparing a fibrinsponge based on fibrin, which is characterized by the following steps:

preparing a fibrin clot by mixing fibrinogen and thrombin and incubatingthe mixture at a temperature, preferably at 20 to 37° C., for a periodof time sufficient for the formation of the fibrin, clot and inparticular to complete cross-linking of the fibrin, preferably 10 min to24 h,

(deep-freezing and) lyophilizing the fibrin clot, and

adjusting a residual moisture of at least 3%, preferably 3 to 35%, inparticular between 10 and 200%.

In a preferred embodiment, adjusting of the water content is effectedduring lyophilizing. The residual moisture may, however, also beadjusted to the desired level after lyophilisation by incubating in ahumid chamber.

When producing a preparation according to the invention, preferably alsoa washing and/or incubating step is provided. By this, in particularsoluble components, such as e.g. salts, can be removed, while thecontent of thrombin which is given on account of the preparation methodsurprisingly is retained to a large extent.

Washing may be effected with distilled water, yet also solutionscontaining further active substances or additives may be used. Thus itis possible to purposefully adjust or vary the composition of the finalproduct.

In particular it has been found that the thrombin contained in thefibrin clot practically cannot be eluted by washing with an isotonicsaline solution. For instance, after washing for 4 hours with the10-fold volume of isotonic saline solution, only approximately 4% of thetotal thrombin are found in the washing solution. On the other hand,other plasma proteins, such as, e.g. albumin, are eluted comparativelyrapidly under the same conditions (approximately 25% in 4 hours,approximately 50% in 8 hours).

Finally, the as such surprisingly high stability of the blood clottingactivator or proactivator, respectively, which preferably ishomogenously distributed in the preparation according to the invention,can still be further increased by stabilizers known per se, such as,e.g., sugars, sugar alcohols, polyoles, amino acids, etc., or mixturesthereof.

In a further preferred embodiment, in the course of its preparation, thefibrin sponge is washed and/or impregnated with further substances, ashas already been mentioned earlier. Preferably, the fibrin clot isimpregnated prior to lyophilisation. It is, however, also possible toeffect washing or incubating, respectively, after lyophilisation, thisthen being followed by a further lyophilisation step.

The fibrin foam can be prepared by foaming the fibrinogen solutionbefore or immediately after mixing with thrombin, yet before clottingoccurs. All the means common in the prior art may be utilized for thisfoaming. For instance, these may be chemical, physical and/or mechanicalmeans or methods. Gases may be introduced or generated, in particularair is introduced by intesive, mechanical stirring (Ultraturraxtreatment or the like). As an example of a physical method, theapplication of a vacuum may be mentioned.

Furthermore, however, it has been found that in contrast to the hithertodescribed methods of preparing fibrin sponges, it is not absolutelynecessary to foam the fibrinogen solution or the fibrinogen thrombinmixture before the onset of clotting.

In contrast to the hitherto described methods of preparing fibrinsponges, particularly fine-porous and absorbent preparations areproduced after lyophilisation without substantial shrinkage from fibrinclots that have been prepared without foaming.

Before freezing and lyophilizing, the fibrin clot preferably has arelatively low salt content or a low ionic strength, respectively,preferably less than 0.15, preferably it is substantially free fromsalts.

Avoiding foaming substantially simplifies and facilitates thepreparation method, particularly if the latter is to be carried outunder sterile conditions.

In a further preferred embodiment, the fibrin sponge, i.e. in particularthe fibrin flat material or the fibrin foam, is cut after its respectivepreparation. Preferably, it is cut such that after cutting, the cut areawhich then can be used as a wound cover is as large as possible.Surprisingly it has been found that the absorptive capacity of these cutareas is even higher than that of the uncut, so-to-speak “native”,surfaces immediately after lyophilisation.

The good tolerance even after multiple application, in particular theavoidance of allergic reactions, is preferably attained by the exclusiveuse of human proteins as well as by avoiding methods, e.g. sterilizingmethods, by which the proteins may become denatured (such as, e.g., byγ-irradiation)

In a particularly simple embodiment, the preparation of the wound coveraccording to the invention is effected from a sterile starting material,by maintaining sterile conditions up to the final product.

Preferably, the preparation according to the invention is prepared fromhuman plasma protein fractions which have been treated according tosuitable virus inactivation methods for sensitive plasma proteins.

The inactivation treatment preferably is ensured by a tenside and/orheat treatment, e.g. by a heat treatment in the solid state, inparticular a vapour treatment according to EP 0 159 311, EP 0 519 901 orEP 0 674 531.

In one preferred embodiment the heat treatment is carried out as avapour heat treatment with a residual water content between 6 and 15%,preferably about 12%. When thrombin is present in the sponge it wassurprisingly found that even under these conditions nearly all of thethrombin activity was maintained.

Further steps for inactivating viruses also comprise the treatment withchemical or chemical/physical methods, e.g. with chaotropic substancesaccording to WO 94/13 329, DE 44 34 538 or EP 0 131 740 (solvents) orphotoinactivation.

Preferably, two independent virus inactivation methods are carried out.

Nanofiltration also constitutes a preferred method for depleting viruseswithin the scope of the present invention.

A further possibility of increasing the virus safety consists ininitially starting the preparation exclusively from—to the greatestpossible extent—virus-safe starting substances and then to exclude allrisks of contamination in the course of the preparation process. Thegreatest possible safety in terms of the transmission of viral or otherpathogens in the preparation from human plasma is additionally ensuredby carefully selecting and monitoring the plasma donors, testing thestarting plasma for pathogenic viruses (preferably single donationtesting), in particular by means of PCR methods.

Yet basically, the preparation according to the invention may also beprepared from recombinant or transgenic material, wherein the necessarysafety measures also have to be met.

Preferably, the preparation is effected under sterile conditions,starting from sterile intermediate products (sterile-filtered solutions)up to the sterile packed final product, so that a subsequentsterilisation in the final container, such as, e.g., by γ-irradiation,is no longer necessary.

The complete absorption during the wound healing process as well as thesmooth and undisturbed wound healing are attained by using fibrin andoptionally further plasma proteins, such as, e.g., fibronectin, as thematrix, the formation of fibrin preferably taking place at approximatelyphysiologic ionic strength, at approximately physiologic pH and in thepresence of factor XIII, so that a cross-linked fibrin clot having aphysiological fibrin structure is obtained which is essential for thegermination of fibroblasts and thus for a rapid and smooth wound healing(cf. also Redl at al. Med. Welt, 36, pp. 769-776, 1985).

The length and width of the fibrin sponge according to the invention canbe chosen freely depending on the type of use. Its thickness ispreferably in a practicable range of between 1 and 20 mm, depending onthe indication, thicknesses between 3 and 15 or between 5 and 10 mm,respectively, being preferred.

The fibrin sponge according to the invention may also be present as acombination with other materials.

In a specific embodiment the sponge is made up of several layers so thata multilayered material is formed. Preferably a least one layer hashemostatic properties. The layers may be built up by the same materialor they may differ in material and/or their composition.

The sponge material may be characterized by the specific weight of thematerial or each layer. In a preferred embodiment the specific weight isbetween 0.005 and 0.15 g/cm³, more preferably 0.01 to 0.09 g/cm³, mostpreferred 0.02 to 0.05 g/cm³.

In another preferred embodiment the layers are composed of the samematerial but differing in their physical properties like e.g. theirspecific weight. By varying the physical properties of the layers, asponge may be obtained which is characterized in that one side of thissponge is smooth, with a high absorptive capacity, while the other sideof the sponge is more compact and tight. Another way of varying theproperties of the different layers can be achieved by varying the amountor concentration of the substances forming the layer or of thosesubstances with which each layer is impregnated. So for example, theconcentration of thrombin in the different layers may be different.

The use of the fibrin foam, e.g., will be such that it is applied ontothe bleeding wound and is slightly pressed thereto for a short period oftime (approximately 30 seconds). The sponge sucks up the blood veryrapidly, the sucked up blood clotting within the sponge. Thereby a tightanchoring of the fibrin sponge on the wound surface will be attained,which at last is the basis of its hemostatic and adhesive effect.

The preparation may also be used as a so-called dry adhesive, preferablyas a relatively thin sponge. To glue together two (soft) bleeding tissueparts, a suitable piece of the “dry adhesive” is applied to a woundsurface, and subsequently the second wound surface (the second tissuepart) is adapted and shortly pressed thereto.

The medical field of indication for the fibrin sponge according to theinvention is quite large. Not only can the sponge be used to stop aseeping hemorrhage, it may also be used for stopping a hemorrhage incase of very large bleeding areas with a high blood pressure. Thefollowing internal and external surgical procedures may successfully becarried out by using the fibrin sponge according to the invention:general surgery, e.g. surgery on parenchymatous organs (liver, kidney,spleen etc.), cardiovascular surgery, thorax surgery, grafting surgery,orthopedic surgery, surgical operations in the fields of bone surgeryand plastic surgery, ear, nose and throat surgery, operations in thefield of neurosurgery, operations in the urologic and gynecologictracts, as well as generally for hemostasis as well as for treatingconventional wounds.

According to a further aspect of the present invention, also a kit forgluing wounds is provided, which is characterized in that it comprises afibrin sponge according to the invention and a component containing ablood clotting factor.

Preferably, this blood clotting factor is fibrinogen, factor XIII,fibronectin, thrombin or mixtures of these factors, a fibrinogencomponent preferably in storage-stable form, such as a commerciallyavailable product of a tissue adhesive based on fibrinogen beingparticularly preferred.

The sponge according to the present invention may also be useful as abiological support for cell cultures, especially for mammalian cellslike e.g. keratinocytes, epithelial cells, epidermal cells, fibroblasts,chondrocytes or the like.

Therefore, according to the present invention also a kit is providedcomprising a fibrin sponge according to the present invention in sterileform for use as a biological support for cell cultures.

Further additives like e.g. antibiotics, various amino acids and/orvarious growth factors may be present within the sponge or may be addedlater to the used medium.

In using the fibrin sponge according to the present invention as asupport for growing cells, it is possible to form replacements of tissuelike e.g. skin, bone, cartilage and/or nerves, respectively.

The invention will be explained in more detail by the following examplewithout, however, restricting it thereto:

EXAMPLE 1

Fibrin fleece-like flat material of cross-linked fibrin having a(relatively low) content of thrombin.

1.1 Preparation

A commercially available tissue adhesive preparation, virus inactivatedby vapour treatment and lyophilized (Tissucol®, IMMUNO AG) served as thestarting material for preparing a fibrinogen solution having a contentof factor XIII and fibronectin. The preparation was dissolved accordingto the producer's instructions with an aqueous aprotinin solution (3000KIU/ml) and further diluted 1:4 with distilled H₂O to yield a fibrinogenconcentration of approximately 20 mg/ml. 20 ml of this solution wererapidly mixed with 20 ml of a thrombin-CaCl₂ solution (human thrombin,approximately 10 U/ml 5 mM CaCl₂) and poured into a Petri dish (ID=8.4cm), at first allowed to stand undisturbed at room temperature forapproximately 30 min and then incubated for approximately 16 furtherhours in a humid chamber at 37° C. The clot in form of a round dischaving a thickness of approximately 7 mm was then deep-frozen andlyophilized. The lyophilisate formed (fleece-like flat material) had alight, fine-porous appearance and substantially retained the form of theclot prior to lyophilisation. Yet the material was still too brittle foran optimum application. To improve the desired softness and pliability,the flat material was incubated for approximately 3 h in a humid chamberat room temperature, whereby a soft, adaptable and highly absorbentmaterial was obtained.

1.2: Analysis, in vitro testings: Residual moisture: approximately 15%Pliability: >90° at a mean radius of curvature corresponding to half thelayer thickness Fibrin cross-linking: γ-chains: 100% α-chains:approximately 80% Thrombin content: determination after extraction with1 M NaCl: 1.4 U/cm³ Water absorption capacity: approximately the 6-foldof its own weight Water absorption rate: a drop of water (50 μl) placedonto the surface of the flat material is completely sucked up inapproximately 25 s

(on cut areas of the flat material under otherwise equal conditionswater is absorbed practically immediately, i.e. in less than 5 s).

1.3 Efficacy

The adhesive and hemostatic effect of the preparation prepared accordingto 1.1 was tested in the so-called kidney pole resection model on arabbit.

For this, a kidney was exposed on the test animal under anesthesia withkedamin and xylazin (after-dosing with pentobarbital), and the kidneypole was removed with the scalpel. A circular area bleeding with uniformintensity and having a diameter of approximately 1 to 1.5 cm is formed.The test preparation is applied onto the bleeding area, slightly pressedthereto by means of the finger for exactly 30 s and then released.Adhesion of the preparation is observed, and the time until completehemostasis is measured. Possibly seeped out blood is taken up with gauzeswabs, and the loss of blood is calorimetrically determined afterextraction with ammonia from the test material and the gauze swabs.

Testing with the preparation obtained according to 1.1. was performed ontwo rabbits.

Results

In both instances, the preparation adhered well on the intensivelybleeding surface, and complete hemostasis was attained practicallyimmediately (in less than 30 s). No after bleedings occurred during asecondary observation period of 1 h.

Total blood loss (sucked up in the test material, no other seeped outblood) amounted to 0.10 and 0.18 ml, respectively.

In comparison thereto, with conventional hemostasis by means auf gauzethe blood loss in this model is approximately 5 ml with a bleeding timeof 3 min, and approximately 27 ml with a bleeding time of 9 min withoutany treatment.

The substantially improved efficacy of the preparation obtainedaccording to 1.1 as compared to conventional means thus has beenimpressively demonstrated.

What is claimed is:
 1. A storage-stable fibrin sponge comprising aresidual moisture of at least 10%.
 2. The storage-stable fibrin spongeas set forth in claim 1, wherein said residual moisture ranges from 10%to 35%.
 3. The storage-stable fibrin sponge as set forth in claim 1,wherein said residual moisture ranges from 10 to 20%.
 4. Thestorage-stable fibrin sponge as set forth in claim 1, further comprisinga blood clotting activator or proactivator.
 5. The storage-stable fibrinsponge as set forth in claim 4, wherein said blood clotting activator orproactivator is selected from the group consisting of thrombin,prothrombin, activated factor X, activated prothrombin complex, FEIBA,calcium ions and mixtures thereof.
 6. The storage-stable fibrin spongeas set forth in claim 5, wherein said blood clotting activator isthrombin, said thrombin being comprised in an amount of from 1 to 300U/cm³.
 7. The storage-stable fibrin sponge as set forth in claim 5,wherein said thrombin is comprised in an amount of from 5 to 100 U/cm³.8. The storage-stable fibrin sponge as set forth in claim 5, whereinsaid thrombin is comprised in an amount of from 10 to 50 U/cm3.
 9. Thestorage-stable fibrin sponge as set forth in claim 1, further comprisinga member selected from the group consisting of: stabilizers,preservatives, antibiotics, therapeutic agents, antifibrinolytic agents,growth factors, further plasma proteins, enzymes, inhibitors andmixtures of these agents.
 10. The storage-stable fibrin sponge as setforth in claim 1, said fibrin sponge having a liquid absorption capacityof at least the 2-fold of its own weight.
 11. The storage-stable fibrinsponge as set forth in claim 1, wherein said fibrin sponge has a liquidabsorption capacity which is 4-fold of its own weight.
 12. Thestorage-stable fibrin sponge as set forth in claim 1, wherein saidfibrin sponge is virus inactivated.
 13. The storage-stable fibrin spongeas set forth in claim 12, wherein said virus inactivation has beeneffected in two independent virus inactivation steps.
 14. Thestorage-stable fibrin sponge as set forth in claim 1, said fibrin spongebeing free from elutable plasma proteins.
 15. The storage-stable fibrinsponge as set forth in claim 1, said fibrin sponge being free fromelutable thrombin.
 16. The storage-stable fibrin sponge as set forth inclaim 1, said fibrin sponge having been prepared from virus-safestarting substances.
 17. The storage-stable fibrin sponge as set forthin claim 1, said fibrin sponge being provided in an aseptic package andin ready-to-use form.
 18. The storage-stable fibrin sponge as set forthin claim 1, said fibrin sponge having a thickness of at least 1 mm. 19.The storage-stable fibrin sponge as set forth in claim 1, said fibrinsponge having a thickness of between 5 and 10 mm.
 20. The storage-stablefibrin sponge as set forth in claim 1, said fibrin sponge beingmultilayered.
 21. The storage-stable fibrin sponge as set forth in claim20, wherein at least one layer of said multilayered fibrin sponge hashemostatic properties.
 22. The storage-stable fibrin sponge as set forthin claim 1, said fibrin sponge having a specific weight of between 0.005and 0.15 g/cm3.
 23. The storage-stable fibrin sponge as set forth inclaim 22, wherein said specific weight of said fibrin sponge rangesbetween 0.01 and 0.09 g/cm3.
 24. The storage-stable fibrin sponge as setforth in claim 22, wherein said specific weight of said fibrin spongeranges between 0.02 and 0.05 g/cm3.
 25. A kit for wound coverage, saidkit comprising a storage-stable fibrin sponge having a residual moistureof at least 10% and a component containing a blood clotting factor. 26.The kit as set forth in claim 25, wherein said blood clotting factor isselected from the group consisting of fibrinogen, factor XIII,fibronectin, thrombin and mixtures of said factors.
 27. A method ofpreparing a fibrin sponge, said method comprising preparing a fibrinclot by mixing a fibrinogen solution with a thrombin solution andincubating the resulting mixed solution at a certain temperature for aperiod of time sufficient for formation of said fibrin clot, b)deep-freezing and lyophilizing said fibrin clot, and c) adjusting aresidual moisture in said fibrin clot to at least 10%.
 28. A method asset forth in claim 27, wherein said fibrinogen solution contains factorXIII, and said thrombin solution contains Ca²+ ions, and saidtemperature for incubating ranges between 20 and 37° C., and incubatingof said mixed solution is carried out for a period of time sufficientfor cross-linking of said fibrin formed, and wherein a residual moistureof from 10% to 35% is adjusted.
 29. The method as set forth in claim 27,wherein said incubating of said mixed solution is carried out for aperiod of time of from 10 min to 24 h, and wherein a residual moistureof from 10 to 20% is adjusted.
 30. The method as set forth in claim 27,further comprising packing said fibrin sponge in a suitable container.31. The method as set forth in claim 30, wherein said container is atleast one of air-tight and water-tight.
 32. The method as set forth inclaim 27, wherein at least one of said fibrinogen solution and saidthrombin solution further comprises at least one of another plasmaprotein, another active substance and an auxiliary agent.
 33. The methodas set forth in claim 32, further comprising at least one of washingsaid fibrin clot with water or with an aqueous solution and incubatingsaid fibrin clot with an additional solution, said additional solutioncontaining at least one member selected from the group consisting ofthrombin, another plasma protein and another active substance.
 34. Themethod as set forth in claim 33, wherein said at least one of washingand incubating with said further solution containing said at least oneof thrombin, an additional plasma protein and an auxiliary agent iseffected after lyophilizing, followed by another deep-freezing andlyophilizing step.
 35. The method as set forth in claim 32, wherein saidadditional plasma protein is selected from the group consisting offibronectin, a fibrinolysis inhibitor, plasminogen and albumin, andwherein said additional active substance is selected from the groupconsisting of a non-plasmatic and synthetic fibrinolysis inhibitor, anantibiotic and a growth hormone.
 36. The method as set forth in claim32, wherein said auxiliary agent is selected from the group consistingof sugar, sugar alcohol, polyol, amino acid, tenside, plasticizer and amoistening agent.
 37. The method as set forth in claim 32, wherein saidauxiliary agent is glycerol.
 38. The method as set forth in claim 27,further comprising foaming said fibrinogen solution or a mixture of saidfibrinogen solution with said thrombin solution before said formation ofsaid fibrin clot.
 39. The method as set forth in claim 27, furthercomprising cutting said fibrin clot after said fibrin clot has beenlyophilized.
 40. The method as set forth in claim 27, wherein saidpreparing of said fibrin clot is effected from a sterile startingmaterial, further comprising maintaining sterile conditions throughoutsaid method.
 41. A method of treating a patient suffering from woundsand hemorrhages, said method comprising applying to said patient afibrin sponge having a residual moisture of at least 10%, for at leastone of hemostasis, tissue adhesion, cell culture supported healing andwound healing.
 42. A kit comprising a storage-stable fibrin spongehaving a residual moisture of at least 10% is sterile form for use as abiological support for cell cultures.